Monoclonal antibodies have proven useful as reagents for research, and also as clinical agents for both therapy and diagnosis. Human monoclonal antibodies are particularly useful for this purpose. The introduction of phage display technology has provided a tool for the generation of human antibodies which circumvents the limitations of earlier antibody generating technologies. Phage display technology made it possible to generate large antibody repertoires within E. coli. The antibody repertoire is expressed in the bacteria, and subsequently exposed to a selective pressure to thereby obtain desired antibody characteristics. Several other in vitro antibody display methods, such as ribosomal display and bacterial, yeast and mammalian cell surface display, have also emerged and also allow the production of diverse antibody libraries.
The antigen-binding site of an antibody typically comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). The majority of the diversity in the antigen specificity of an antibody is provided by six loops or regions, known as the complementary determining regions (CDRs), with three CDRs present in the heavy chain variable domain (the VH CDRs 1, 2 and 3) and three CDRs present in the light chain variable domain (the VL CDRs 1, 2 and 3). All six CDRs of an antibody, supported by more conserved framework regions, constitute a functional antigen-binding site. VH CDR3 and VL CDR3 are naturally the most diverse and are therefore considered to be the most important for antigen recognition. The VH and VL CDRs 1 and 2 are considered to have a more subordinate role in antigen recognition.
A synthetic antibody library comprises designed diversity primarily in the CDRs, which is introduced by controlled synthesis of the genes encoding the variable domains. The earliest synthetic antibody libraries were semi-synthetic, comprising CDRs from natural sources with designed variations introduced in parts. Hoogenboom and Winter used in their design of a semi-synthetic scFV antibody library a variety of different framework genes in combination with diversity generated by randomization of positions in the VH CDR3 region (Hoogenboom and Winter, J Mol Biol. 1992 Sep. 20; 227(2):381-8). The synthetic design was expanded to length variations of CDR3. Residues in VL CDR3 as well as in the VH CDR3 were randomized. The added features of the synthetic design generated antibody libraries of increased size.
A fully synthetic library design was demonstrated by Sidhu et al., who applied a restricted design to the CDRs (Sidhu et al., J. Mol. Biol. 2007, 373, 924-940). The diversity was restricted to the binary code of tyrosine and serine in VH CDR1 and VH CDR2. The VL CDR3 was restricted to the binary code of tyrosine and serine while the VH CDR3 was allowed chemical complexity. The amino acid composition of VH CDR3 was biased for tyrosine, serine, and glycine, while allowing all other amino acids (19) except for cysteine which was excluded. The restricted design produced highly functional phage-displayed libraries.
The demand for novel high affinity antibodies for clinical uses remains high. Thus, there remains a need for diverse libraries of antibody molecules. This need is met by the present invention, which also provides other advantages.